In a state referred to as [PSI+], the yeast protein Sup35p folds abnormally causing it to aggregate. [PSI+] propagates in the cytoplasm as a conformationally altered Sup35p in a manner consistent with the prion hypothesis. We use [PSI+] as model for studying prions and other amyloidogenic proteins. It is normally transmitted infallibly during mitosis and meiosis. By an unknown mechanism, [PSI+] replication is blocked by millimolar amounts of guanidine hydrochloride (Gdn-HCl) in growth media causing cells to lose [PSI+]. The protein chaperone Hsp104 is required for prion replication in yeast but how it acts in this process is unclear. In a search for cellular functions affecting [PSI+] we isolated a mutant allele of an Hsp70 protein chaperone. This led to the discovery that cytosolic Hsp70 provides an apparently essential activity in [PSI+] propagation by facilitating the generation of new prion particles from preexisting material. We also discovered that the presence of [PSI+] causes a stress to cells that derepresses expression of heat shock proteins. Two mutant alleles of HSP104 that adversely affect [PSI+] were also identified. One mutant has been found to have a novel alteration in an evolutionarily conserved residue and may provide clues as to how Hsp104 acts in [PSI+] replication. I have also discovered that in related non-isogenic strains, certain combinations of Hsp70 mutations cause high loss of [PSI+] during meiosis. In some others, meiotic segregation of [PSI+] is normal but [PSI+] is rapidly lost from spores as they age. Isogenic strains are being constructed to verify and then characterize these effects. I have discovered that addition of Gdn-HCl to growing cells significantly impairs Hsp104 function without reducing its abundance, indicating that Hsp104 is a target of Gdn-HCl. Since [PSI+] replication is dependent on Hsp104, this may explain how Gdn-HCl cures [PSI+]. Repeated isolation of mutations in Hsp70 and Hsp104 underscores their importance in [PSI+] propagation. Continued study of these chaperones using our system is expected to provide additional insight into the molecular mechanisms underlying their roles in prion replication.Publications in the last calendar year:Jung, G., Jones, G. W., Wegrzen, R. W., and Masison, D. C. (2000). A role for cytosolic Hsp70 in yeast [PSI+] prion propagation and [PSI+] as a cellular stress. Genetics 156: in press. (October)Masison, D. C. (2000). Expanding the prion model for the yeast [PSI+] element. Trends Microbiol. 8: 1-2. (Commentary)